The cloning and expression of heterologous genes in fungi has been used to produce a variety of useful proteins. For example: Lambowitz, U.S. Pat. No. 4,486,533, discloses the autonomous replication of DNA vectors for filamentous fungi by mitochondrial plasmid DNA and the introduction and expression of heterologous genes into Neurospora; Yelton et al., U.S. Pat. No. 4,816,405, discloses tools and systems that enable the modification of important strains of filamentous ascomycetes to produce and secrete large quantities of desired heterologous proteins; Buxton et al., U.S. Pat. No. 4,885,249, discloses the transformation of Aspergillus niger by a DNA vector that contains a selectable marker capable of being incorporated into the host A. niger cells; and McKnight et al., U.S. Pat. No. 4,935,349, discloses a method for expressing higher eukaryotic genes in Aspergillus involving promoters capable of directing the expression of a heterologous gene in Aspergillus and other filamentous fungi. Similar techniques have been used to clone the mtr gene involved with amino acid transport in Neurospora crassa (“N. crassa”) and to verify the tight linking of the cloned DNA to genomic markers flanking this gene in vivo. Stuart, W. D. et al., Genome (1988) 30:198-203; Koo, K. and Stuart, W. D. Genome (1991) 34:644-651.
Filamentous fungi possess many characteristics which make them good candidates for use in producing eukaryotic proteins. Filamentous fungi can secrete complex proteins; correctly fold three dimensional proteins including disulfide bond formation; proteolytically clip proteins following translation; and glycosylate proteins using n-linked and o-linked glycosylation reactions. These abilities have made this group of organisms attractive hosts for the production of secreted recombinant proteins. (MacKenzie, D. A. et al., J Gen Microbiol (1993) 139:2295-2307; Peberdy, J. F., Trends in BioTechnology (1994) 12:50-57).
Neurospora crassa has been used as a host cell for recombinant homologous and heterologous protein production. (Carattoli, A., et al., Proc Nat Acad Sci USA (1995) 92:6612-6616; Yamashita, R. A. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A; Kato, E. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A; Buczynski, S. et al. Fungal Genetics Newsletter (1995 Suppl.) 42A, Nakano, E. T. et al. Fungal Genetics Newsletter (1995 Suppl.) 40:54 ( )). In addition, Neurospora crassa has recently been used as a host cell for expressing recombinant heterodimeric and multimeric proteins by means of a heterokaryon., PCT Application WO 95/21263.
A “heterokaryon” (or a heterokaryonic cell) is a cell formed from the fusion of two filamentous fungal parent strains, each heterokaryon cell thus containing two (or more) genetically different nuclei. Heterokaryons contain nuclei from two parent strains that are generally homozygous for all heterokaryon compatibility alleles (except for the mating type allele when the tol gene is present). At least ten chromosomal loci have been identified for heterokaryon incompatibility: het-c, het-d, het-e, het-i, het-5, het-6, het-7, het-8, het-9 and het-10, and more are inferred to exist. Perkins et al, “Chromosomal Loci of Neurospora crassa”, Microbiological Reviews (1982) 46:426-570, at 478.
The present invention advances the work of that disclosed in PCT Application WO 95/21263 by providing methods and compositions for producing a population of multimeric proteins using heterokaryonic filamentous fungi. Such methods and compositions are useful in the discovery and production of panel of multimeric molecules, such as a heterodimeric antibody, multimeric hormones and growth factors and multimeric receptors.